This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We propose to elucidate the structural architecture and the catalytic mechanisms of the mitochondrial machinery responsible for the biosynthesis of iron-sulfur clusters (ISC). ISC are highly versatile co-factors and cells utilize ISC-containing enzymes in a variety of capacities throughout the cell. Yeast cells carry out ISC biosynthesis primarily in the mitochondrial matrix while mammalian cells synthesize Fe-S clusters in both mitochondria and cytosol. However, problems in human cells with mitochondrial ISC biogenesis adversely affect cytosolic iron status and ISC biogenesis. Consequently, a complete loss of mitochondrial ISC biosynthesis is incompatible with life. Moreover, partial losses result in severe phenotypes dominated by impaired energy metabolism, elevated mitochondrial iron levels, iron-dependent oxidative damage, and loss of mitochondrial DNA integrity, concomitantly with cytoplasmic iron depletion and non-mitochondrial enzyme deficits leading to nuclear genome instability among other effects. Inherited defects in mitochondrial ISC biosynthesis have thus far been associated with the neurodegenerative disease Friedreich ataxia and with tissue-specific conditions including myopathy and sideroblastic anemia. Our limited mechanistic understanding of this process hampers our ability to interrogate its functionality directly in normal conditions and disease states. Approach. Our work suggests that in yeast and human mitochondria, ISC biosynthesis occurs on stable complexes made of multiple copies of at least three components (iron- donor, sulfur-donor, and scaffold) reaching molecular masses of megadaltons. The protein- protein interaction surfaces, catalytic mechanisms and overall architecture of these macromolecular machines remain to be described and are the focus of our long-term goals: Long-Term Goal 1: Define the structures of sub-complexes. Long-Term Goal 2: Attempt to define the structure of the whole machinery. Short-Term Goal 1: Show feasibility for 1-3 particularly stable sub-complexes. Short-Term Goal 2: Obtain a subnanometer-resolution 3D reconstruction for one of these three sub-complexes. To better define the protein-protein interaction surfaces and catalytic mechanisms of these complexes, we would like to dock crystal structures into the cryo-EM maps. This will work best with subnanometer maps that reveal secondary structure, which allows for unambiguous determination of each component's position and orientation.